Novel neutrophil chemotactic factor cloned cDNA and monoclonal antibodies thereto

ABSTRACT

An isolated, synthetic preparation of a novel neutrophil-specific chemotactic factor (NCF), monoclonal antibodies having specific binding affinity for NCF and a clone containing the complete cDNA coding sequence for NCF are disclosed.

BACKGROUND OF THE INVENTION

[0001] 1. Technical Field

[0002] The present invention relates to an isolated, syntheticpreparation of a novel neutrophil-specific chemotactic factor (NCF),monoclonal antibodies having specific binding affinity for NCF and aclone containing the complete coding sequence for NCF.

[0003] 2. State of the Art

[0004] Activated monocytes/macrophages produce various mediators thatcause inflammation. Among them are chemotactic factors which cause whiteblood cells to migrate into inflammatory sites where these factors arereleased. Neutrophils, the dominant leukocytes attracted by thechemotactic factors, are believed to play a critical role in theinflammatory reactions. Such diseases as rheumatoid arthritis,idiopathic pulmonary fibrosis and certain pathological inflammatorychanges in many other conditions are believed to be caused byneutrophils and/or their products. However, a specific pro-inflammatorymediator released by tissue macrophages and other cells in response toinflammatory stimuli and leading to neutrophil-rich leukocyteaccumulation in host defense and disease, has not heretofore beenidentified and isolated.

SUMMARY OF THE INVENTION

[0005] It is, therefore, an object of the present invention to provide abiologically active novel synthetic polypeptide acting as aneutrophil-specific chemotactic factor (NCF).

[0006] It is a further object of the present invention to provide amolecular clone containing the complete coding sequence for thesynthesis of NCF by either prokaryotic or eukaryotic expression vectors.

[0007] It is a still further object of the present invention to providemonoclonal antibodies having specific binding affinity for NCF of thepresent invention.

[0008] It is another object of the present invention to provide a kitcomprising a container containing the cDNA for NCF quantitation,detection or localization of NCF mRNA in a body sample.

[0009] It is yet another object of the present invention to provide akit comprising a container containing anti-NCF antibodies havingspecific binding affinity for NCF for quantitation, detection orlocalization of NCF in a body sample.

[0010] Other objects and advantages will become evident from thefollowing detailed description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011] These and other objects, features and many of the attendantadvantages of the invention will be better understood upon a reading ofthe followings detailed description when considered in connection withthe accompanying drawings wherein:

[0012]FIG. 1 demonstrates translation of cDNA into NCF protein inreticulocyte lysate system; and

[0013]FIG. 2 shows:

[0014] (a) Northern blot analysis of mRNA induction inlipopolysaccharide (LPS) stimulated peripheral blood monomuclear cells(PBMC);

[0015] (b) The time course of the accumulation of neutrophil chemotacticactivity in culture media of PBMC after stimulation with LPS;

[0016] (c) Induction of NCF mRNA in PBMC by IL 1 or TNF, but not by IL2, gamma-IFN, and alpha-IFN;

[0017] (d) HPLC gel filtration analysis of IL 1 and TNF inducedneutrophil chemotactic activity.

DETAILED DESCRIPTION OF THE INVENTION

[0018] The above and various other objects and advantages of the presentinvention are achieved by a homogeneously pure, isolated, syntheticneutrophil chemotactic protein, designated herein NCF, composed in thewhole or in part only of the following amino acid sequence (singleletter code): NH₂-S-A-K-E-L-R-C-Q-C-I-K-T-Y-S-K-P-F-H-P-K-F-I-K-E-L-R-V-I-E-S-G-P-H-C-A-N-T-E-I-I-V-K-L-S-D-G-R-E-L-C-L-D-P-K-E-N-W-V-Q-R-V-V-E-K-F-L-K-R-A-E-N-S

[0019] Unless defined otherwise, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Although any methodsand materials similar or equivalent to those described herein can beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedhereunder are incorporated herein by reference. Unless mentionedotherwise, the techniques employed herein are standard methodologieswell known to one of ordinary skill in the art.

[0020] Chemical synthesis of the NCF of the present invention composedof the 72 amino acid residues as shown above, is achieved bycommercially available polypeptide synthesizers. Alternatively, the NCFof the present invention is synthesized by standard techniques employingan expression vector containing in its genome the cloned complete codingsequence of NCF. Anti-NCF monoclonal antibodies of the present inventionare prepared by standard hybridoma technology and utilized forpurification and assaying purposes following standard immunologicalmethodologies well known in the art.

[0021] High performance liquid chromatography (HPLC), in situhybridization assays, Northern blotting analysis and the like aretypical examples of the standard conventional techniques well known toone of ordinary skill in the art, which can be employed for isolation,localization, differentiation, detection, or measurement of the mRNA forNCF in biological samples.

[0022] It should be noted that the fact that chemically synthesizedpolypeptide of the present invention at 10 factor, is shown by theresults presented in Table 1. TABLE 1 Chemotactic response of humanneutrophils to chemically synthesized NCF. Concentration of Percentageof assay NCF, nanomolar neutrophils that migrated 1000 23  100 34  10 32  1 5   0.1 1 Hanks medium 0.3 10-⁷M fMet-Leu-Phe 40

[0023] It may be pointed out that various stimuli cause the release orsecretion of more than one chemoattractant. Without the cDNA of thepresent invention, it is clear, of course, that the presence,specificially of the mRNA for NCF as an involved factor in a particularclinico-pathological situation, could not be definitively identified anddiagnosed. cDNA of the present invention due to its binding affinity formRNA for NCF, for the first time makes it possible to analyze bodysamples such as joint fluid, sputum, alveolar lavage fluid, tissuesamples and the like to detect the presence or absence of mRNA for NCF.Of course, the antibodies can also be utilized for diagnostic purposesto detect the NCF and to neutralize the NCF for alleviating any diseaseor anomalous conditions in which the presence of NCF is found to be acausative factor.

[0024] A pharmaceutical composition for use in treating inflammatorycondition comprises and anti-inflammatory effective amount of theanti-NCF monoclonal antibodies in pharmaceutically acceptable carrier,such as physiological saline, sterile non-toxic buffer and the like.

[0025] A deposit of cDNA for NCF and of the hybridoma for anti-NCFmonoclonal antibodies have been made at the ATCC, Rockville, Md. on Jan.12, 1988 and Feb. 17, 1988, respectively, under the accession numbers40412 and HB9647, respectively. The deposits shall be viably maintained,replacing if they became non-viable, for a period of 30 years from thedate of the deposit, or for 5 years from the last data of request for asample of the deposit, whichever is longer, and made available to thepublic without restriction in accordance with the provisions of the law.The Commissioner of Patents and Trademarks, upon request, shall haveaccess to the deposits.

[0026] It is understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication and scope of the appended claims.

1 1 1560 BASE PAIRS NUCLEIC ACID UNKNOWN UNKNOWN 1 CTCCATAAGG CACAAACTTTCAGAGACAGC AGAGCACACA 40 AGCTTCTAGG ACAAGAGCCA GGAAGAAACC ACCGGAAGGA 80ACCATCTCAC TGTGTGTAAA CATGACTTCC AAGCTGGCCG 120 TGGCTCTCTT GGCAGCCTTCCTGATTTCTG CAGCTCTGTG 160 TGAAGGTGCA GTTTTGCCAA GG AGT GCT AAA GAA CTT197 AGA TGT CAG TGC ATA AAG ACA TAC TCC AAA CCT TTC 233 CAC CCC AAA TTTATC AAA GAA CTG AGA GTG ATT GAG 269 AGT GGA CCA CAC TGC GCC AAC ACA GAAATT ATT GTA 305 AAG CTT TCT GAT GGA AGA GAG CTC TGT CTG GAC CCC 341 AAGGAA AAC TGG GTG CAG AGG GTT GTG GAG AAG TTT 377 TTG AAG AGG GCT GAG AATTCA TAAAAAAATT CATTCTCTGT 418 GGTATCCAAG AATCAGTGAA GATGCCAGTGAAACTTCAAG 458 CAAATCTACT TCAACACTTC ATGTATTGTG TGGGTCTGTT 498GTAGGGTTGC CAGATGCAAT ACAAGATTCC TGGTTAAATT 538 TGAATTTCAG TAAACAATGAATAGTTTTTC ATTGTACCAT 578 GAAATATCCA GAACATACTT ATATGTAAAG TATTATTTAT618 TTGAATCTAC AAAAAACAAC AAATAATTTT TAAATATAAG 658 GATTTTCCTAGATATTGCAC GGGAGAATAT ACAAATAGCA 698 AAATTGGGCC AAGGGCCAAG AGAATATCCGAACTTTAATT 738 TCAGGAATTG AATGGGTTTG CTAGAATGTG ATATTTGAAG 778CATCACATAA AAATGATGGG ACAATAAATT TTGCCATAAA 818 GTCAAATTTA GCTGGAAATCCTGGATTTTT TTCTGTTAAA 858 TCTGGCAACC CTAGTCTGCT AGCCAGGATC CACAAGTCCT898 TGTTCCACTG TGCCTTGGTT TCTCCTTTAT TTCTAAGTGG 938 AAAAAGTATTAGCCACCATC TTACCTCACA GTGATGTTGT 978 GAGGACATGT GGAAGCACTT TAAGTTTTTTCATCATAACA 1018 TAAATTATTT TCAAGTGTAA CTTATTAACC TATTTATTAT 1058TTATGTATTT ATTTAAGCAT CAAATATTTG TGCAAGAATT 1098 TGGAAAAATA GAAGATGAATCATTGATTGA ATAGTTATAA 1138 AGATGTTATA GTAAATTTAT TTTATTTTAG ATATTAAATG1178 ATGTTTTATT AGATAAATTT CAATCAGGGT TTTTAGATTA 1218 AACAAACAAACAATTGGGTA CCCAGTTAAA TTTTCATTTC 1258 AGATATACAA CAAATAATTT TTTAGTATAAGTACATTATT 1298 GTTTATCTGA AATTTTAATT GAACTAACAA TCCTAGTTTG 1338ATACTCCCAG TCTTGTCATT GCCAGCTGTG TTGGTAGTGC 1378 TGTGTTGAAT TACGGAATAATGAGTTAGAA CTATTAAAAC 1418 AGCCAAAACT CCACAGTCAA TATTAGTAAT TTCTTGCTGG1458 TTGAAACTTG TTTATTATGT ACAAATAGAT TCTTATAATA 1498 TTATTTAAATGACTGCATTT TTAAATACAA GGCTTTATAT 1538 TTTTAACTTT AAAAAAAACC GG 1560

What is claimed is:
 1. An isolated, synthetic neutrophil chemotacticpolypeptide (NCF) composed in whole or in part of the following aminoacid sequence represented by single letter code:NH₂-S-A-K-E-L-R-C-Q-C-I-K-T-Y-S-K-P-F-H-P-K-F-I-K-E-L-R-V-I-E-S-G-P-H-C-A-N-T-E-I-I-V-K-L-S-D-G-R-E-L-C-L-D-P-K-E-N-W-V-Q-R-V-V-E-K-F-L-K-R-A-E-N-S


2. A molecular clone containing the complete cDNA coding sequence forthe synthesis of the NCF of claim 1 when said cDNA is inserted intogenome of an eukaryotic or prokary tic expression vector.
 3. The cloneof claim 2 having identifying characteristics of ATCC
 40412. 4. Ahybridoma producing monoclonal antibodies having specific bindingaffinity for NCF of claim
 1. 5. The hybridoma of claim 4 having theidentifying characteristics of ATCC HB9647.
 6. The anti-NCF monoclonalantibodies having specific binding affinity for NCF of claim
 1. 7 A kitfor determining the level of NCF mRNA present in a sample, comprising acontainer containing cDNA for NCF for preforming assay to datermine thelevel of mRNA for NCF in a biological sample.
 8. A kit for determiningthe level of NCF present in a sample, comprising a container containinganti-NCF antibodies.
 9. A method for detecting the level of mRNA for NCFin a sample, comprising reacting said sample with cDNA and determiningthe occurrence of hybridization by any conventional technique, thepresence of said cDNA-mRNA hybrids being indicative of the presence ofmRNA for NCF in said sample.
 10. A method of treating inflammatorycondition, comprising administering to a host inflicted with aninflammatory condition, anti-inflammatory effective amount of anti-NCFmonoclonal antibodies to alleviate clinico-pathological condition causedby or related to NCF.
 11. A pharmaceutical composition, comprisinganti-inflammatory effective amount of anti-NCF monoclonal antibodies andpharmaceutically acceptable carrier.